The overall objective of this project is to elucidate how replication- dependent and replication-independent histone synthesis are interrelated during the chromosome replication cycle of proliferating cells and in nonproliferating cells. Such knowledge may be useful in developing procedures with selective toxicity for tumor cells by, as one example, disrupting the balance between histone synthesis and DNA synthesis. The following studies are in progress or have been completed this year. (1) In ongoing collaborative studies with Dr. Yves Pommier's group, we are investigating the synthesis, post-translational modifications, and fates of histones during apoptosis. We have found that the histones are stable during this process, but are found in the cytoplasm of apoptotic cells. (2) Screening of cell lines in the DTP Cell Screen has uncovered one cell line that is a natural overproducer of H2AX. We are studying the regulation of H2AX synthesis in this cell line, which may account for about 50% of the total H2A synthesis. (3) Studies on the preparation of mouse cell lines lacking H2AX is progressing with the preparation of the knockout vector. These studies will allow us to differentiate possible functions of H2AX. If H2AX is essential, we may test which of several H2AX genes that have been modified to delete or alter some of the H2AX characteristic's will protect the cell lines from death. If H2AX is not essential we will study the growth characteristics and chromatin of such cell lines. With this approach we will be able to gain insights into the functions that differentiate H2AX from the other H2A subfamilies.